Impaired muscarinic type 3 (M3) receptor/PKC and PKA pathways in islets from MSG-obese rats

Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG β-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K⁺ (P < 0.05). The MSG group also released less insulin in response to 100 μM carbachol, 10 μM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of β-cell dysfunction.     

Anti-caspase-3 preconditioning increases proinsulin secretion and deteriorates posttransplant function of isolated human islets

Human islet isolation is associated with adverse conditions inducing apoptosis and necrosis. The aim of the present study was to assess whether antiapoptotic preconditioning can improve in vitro and posttransplant function of isolated human islets. A dose-finding study demonstrated that 200 μmol/L of the caspase-3 inhibitor Ac-DEVD-CMK was most efficient to reduce the expression of activated caspase-3 in isolated human islets exposed to severe heat shock. Ac-DEVD-CMK-pretreated or sham-treated islets were transplanted into immunocompetent or immunodeficient diabetic mice and subjected to static glucose incubation to measure insulin and proinsulin secretion. Antiapoptotic pretreatment significantly deteriorated graft function resulting in elevated nonfasting serum glucose when compared to sham-treated islets transplanted into diabetic nude mice (p < 0.01) and into immunocompetent mice (p < 0.05). Ac-DEVD-CMK pretreatment did not significantly change basal and glucose-stimulated insulin release compared to sham-treated human islets but increased the proinsulin release at high glucose concentrations (20 mM) thus reducing the insulin-to-proinsulin ratio in preconditioned islets (p < 0.05). This study demonstrates that the caspase-3 inhibitor Ac-DEVD-CMK interferes with proinsulin conversion in preconditioned islets reducing their potency to cure diabetic mice. The mechanism behind this phenomenon is unclear so far but may be related to the ketone CMK linked to the Ac-DEVD molecule. Further studies are required to identify biocompatible caspase inhibitors suitable for islet preconditioning.     

Insulin secretion kinetics from single islets reveals distinct subpopulations

Type II diabetes progresses with inadequate insulin secretion and prolonged elevated circulating glucose levels. Also, pancreatic islets isolated for transplantation or tissue engineering can be exposed to glucose over extended timeframe. We hypothesized that isolated pancreatic islets can secrete insulin over a prolonged period of time when incubated in glucose solution and that not all islets release insulin in unison. Insulin secretion kinetics was examined and modeled from single mouse islets in response to chronic glucose exposure (2.8‐20 mM). Results with single islets were compared to those from pools of islets. Kinetic analysis of 58 single islets over 72 h in response to elevated glucose revealed distinct insulin secretion profiles: slow‐, fast‐, and constant‐rate secretors, with slow‐secretors being most prominent (ca., 50%). Variations in the temporal response to glucose therefore exist. During short‐term (<4 h) exposure to elevated glucose few islets are responding with sustained insulin release. The model allowed studying the influence of islet size, revealing no clear effect. At high‐glucose concentrations, when secretion is normalized to islet volume, the tendency is that smaller islets secrete more insulin. At high‐glucose concentrations, insulin secretion from single islets is representative of islet populations, while under low‐glucose conditions pooled islets did not behave as single ones. The characterization of insulin secretion over prolonged periods complements studies on insulin secretion performed over short timeframe. Further investigation of these differences in secretion profiles may resolve open‐ended questions on pre‐diabetic conditions and transplanted islets performance. This study deliberates the importance of size of islets in insulin secretion. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1059–1068, 2018     

Standardization of insulin secretion from pancreatic islets: validation of a DNA assay

The use of islet DNA content to standardize insulin secretion rates from pancreatic islets of different sizes has been studied. Isolated intact islets were sorted into 4 size categories and perifused with 22 mM glucose, collecting effluent in 5 min fractions for insulin RIA. DNA content of perifused islets was measured by fluorometric assay, and insulin secretion expressed as pmoles/ug DNA/unit time. For islets with diameters less than 300 u (1) insulin secretion was proportional to islet size; (2) insulin release per islet and islet DNA content were strongly correlated; (3) when expressed as a function of DNA content, insulin secretion from different sized islets was not significantly different. These relationships did not continue for very large islets (above 300 u) suggesting a limiting islet size for insulin secretion in vitro. The data demonstrates that expression of insulin secretion from pancreatic islets with diameters less than 300 u, as a function of their DNA content standardizes secretion irrespective of islet size and number, and should allow direct comparison of secretory responses between different islet tissue preparations.     

Effect of fibroblast growth factor and nerve growth factor on the cellular proliferation and functional activity of isolated islands of Langerhans

The effects of fibroblast growth factor (FGF) and nerve growth factor (NGF) on DNA synthesis and insulin secretion were studied in 4-5-day cultures of the isolated neonatal rat islets. FGF (0.1 ng/ml) stimulated significantly the incorporation of 3H-thymidine into DNA of the isolated islets, but failed to change either insulin content in the islets or the rate of insulin secretion. NGF (0.1-1000 ng/ml) did not affect the above parameters. The responses of the islets of Langerhans to increasing concentrations of glucose and isobutylmethylxanthine were not modified after prolonged exposure to NGF. The role of FGF and NGF in the regulation of proliferation and secretory process in pancreatic islet cells is discussed.     

PPARgamma-dependent and -independent effects of rosiglitazone on lipotoxic human pancreatic islets

We explored the in vitro effects of Rosiglitazone (RZG), a PPARγ agonist, on human pancreatic islet dysfunctions induced by chronic free fatty acid exposure. We demonstrated that RZG beneficial effects on insulin secretion and apoptosis did not imply PDX-1 or insulin gene modulation. It rather involved, through a PPARγ-dependent mechanism, a reduction of iNOS overexpressed in lipotoxic islets. This reduction likely led to the restoration of ATP level and insulin secretion as well as the decrease in apoptosis. More interestingly, we also demonstrated that RZG beneficial effects involved PPARγ-independent mechanisms. RZG treatment led to a limitation of oxidative stress exemplified by an increase of GPx and SOD expression. It also increased UCP2 expression that seemed to display antioxidant action in this model. Thus, RZG did not appear to exert a direct action on insulin expression but rather an indirect action on insulin secretion and apoptosis, through PPARγ-dependent and -independent mechanisms, via regulation of nitrogen and oxygen reactive species injury.     

Long-term treatment with atrial natriuretic peptide inhibits ATP production and insulin secretion in rat pancreatic islets

Atrial natriuretic peptide (ANP) levels correlate with hyperglycemia in diabetes mellitus, but ANP effects on pancreatic islet β-cell insulin secretion are controversial. ANP was investigated for short- and long-term effects on insulin secretion and mechanisms regulating secretion in isolated rat pancreatic islets. A 3-h incubation with ANP did not affect basal or glucose-stimulated islet insulin secretion. However, 7-day culture of islets with 5.5 mM glucose and ANP (1 nM - 1 μM) markedly inhibited subsequent glucose (11 mM)-stimulated insulin secretion; total islet insulin content was not affected. Following ANP removal for 24 h, the islet insulin-secretory response to glucose was restored. The insulin-secretory response to other insulin secretagogues, including α-ketoisocaproic acid, forskolin, potassium chloride, and ionomycin were also markedly inhibited by chronic exposure to ANP. However, the combination of potassium chloride and α-ketoisocaproic acid was sufficient to overcome the inhibitory effects of ANP on insulin secretion. The glucose-stimulated increases in islet ATP levels and the ATP/ADP ratio were completely inhibited in ANP 7-day-treated islets vs. control; removal of ANP for 24 h partially restored the glucose response. ANP did not affect islet glycolysis. ANP significantly increased levels of islet activated hormone-sensitive lipase and the expression of uncoupling protein-2 and peroxisome proliferator-activated receptor-δ and -α. Although islet ANP-binding natriuretic peptide receptor-A levels were reduced to 60% of control after 7-day culture with ANP, the ANP-stimulated cGMP levels remained similar to control islet levels. Thus, long-term exposure to ANP inhibits glucose-stimulated insulin secretion and ATP generation in isolated islets.     

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control.

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Taurine supplementation prevents morpho-physiological alterations in high-fat diet mice pancreatic β-cells

Taurine (Tau) is involved in beta (β)-cell function and insulin action regulation. Here, we verified the possible preventive effect of Tau in high-fat diet (HFD)-induced obesity and glucose intolerance and in the disruption of pancreatic β-cell morpho-physiology. Weaning Swiss mice were distributed into four groups: mice fed on HFD diet (36 % of saturated fat, HFD group); HTAU, mice fed on HFD diet and supplemented with 5 % Tau; control (CTL); and CTAU. After 19 weeks of diet and Tau treatments, glucose tolerance, insulin sensitivity and islet morpho-physiology were evaluated. HFD mice presented higher body weight and fat depots, and were hyperglycemic, hyperinsulinemic, glucose intolerant and insulin resistant. Their pancreatic islets secreted high levels of insulin in the presence of increasing glucose concentrations and 30 mM K⁺. Tau supplementation improved glucose tolerance and insulin sensitivity with a higher ratio of Akt phosphorylated (pAkt) related to Akt total protein content (pAkt/Akt) following insulin administration in the liver without altering body weight and fat deposition in HTAU mice. Isolated islets from HTAU mice released insulin similarly to CTL islets. HFD intake induced islet hypertrophy, increased β-cell/islet area and islet and β-cell mass content in the pancreas. Tau prevented islet and β-cell/islet area, and islet and β-cell mass alterations induced by HFD. The total insulin content in HFD islets was higher than that of CTL islets, and was not altered in HTAU islets. In conclusion, for the first time, we showed that Tau enhances liver Akt activation and prevents β-cell compensatory morpho-functional adaptations induced by HFD.     

Reduction of diffusion barriers in isolated rat islets improves survival,but not insulin secretion or transplantation outcome

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter > 150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter &lt; 100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.     

Evidence for secretion of 7B2 by A- and B-cells of hamster pancreatic islets.

7B2 is a neuroendocrine protein, and in the pancreatic islets the presence of 7B2 in A- and B-cells was immunohistochemically demonstrated. In order to examine 7B2 secretion by A- and B-cells of pancreatic islets, we prepared isolated hamster pancreatic islet cells as well as an A-cell-rich culture, and studied 7B2 secretion under certain stimulations. 7B2 was secreted by isolated hamster pancreatic islet cells. This secretion was stimulated by theophylline and arginine, but glucose had a weak effect on the 7B2 secretion. Such a response of 7B2 to the stimulations was different from that of insulin or glucagon. 7B2 secretion was also noted in the A-cell-rich culture. These results suggest that 7B2 is secreted by both A- and B-cells of the hamster pancreatic islets and its secretion is regulated under certain conditions.     

Human and rat amylin have no effects on insulin secretion in isolated rat pancreatic islets

Amylin, an islet amyloid peptide secreted by the pancreatic beta cell, has been proposed as a humoral regulator of islet insulin secretion. Four separate preparations of amylin were tested for effects on hormone secretion in both freshly isolated and cultured rat islets and in HIT-T15, hamster insulinoma cells. With all three experimental models, exposure to human amylin acid and human and rat amylin at concentrations as high as 100 nM had no significant effect on rates of insulin or glucagon secretion. These observations suggest that amylin, even at concentrations appreciably higher than those measured in peripheral plasma, is not a significant humoral regulator of islet hormone secretion.     

PACAP is an islet neuropeptide which contributes to glucose-stimulated insulin secretion

Pituitary adenylate cyclase activating peptide (PACAP) is a ubiquitously distributed neuropeptide which also is localized to pancreatic islets and stimulates insulin secretion. We examined whether endogenous PACAP within the islets might contribute to glucose-stimulated insulin secretion by immunoneutralizing endogenous PACAP. Immunocytochemistry showed that PACAP immunoreactivity is expressed in nerve terminals within freshly isolated rat islets, but not in islets that had been cultured for 48 h. In contrast, islet endocrine cells did not display PACAP immunoreactivity. Addition of either of two specific PACAP antisera markedly inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from freshly isolated rat islets, whereas a control rabbit serum did not affect glucose-stimulated insulin secretion. In contrast, the PACAP antisera had no effect on glucose-stimulated insulin secretion in cultured islets. Based on these results we therefore suggest that PACAP is an islet neuropeptide which is required for the normal insulinotropic action of glucose.     

Dopamine Modulates Insulin Release and Is Involved in the Survival of Rat Pancreatic Beta Cells

The local synthesis of dopamine and its effects on insulin release have been described in isolated islets. Thus, it may be accepted that dopamine exerts an auto-paracrine regulation of insulin secretion from pancreatic beta cells. The aim of the present study is to analyze whether dopamine is a regulator of the proliferation and apoptosis of rat pancreatic beta cells after glucose-stimulated insulin secretion. Glucose stimulated pancreatic islets obtained from male Wistar rats were cultured with 1 or 10 μM dopamine from 1 to 12 h. Insulin secretion was analyzed by RIA. The cellular proliferation rate of pancreatic islets and beta cells was studied with immunocytochemical double labelling for both insulin and PCNA (proliferating cell nuclear antigen), and active caspase-3 was detected to evaluate apoptosis. The secretion of insulin from isolated islets was significantly inhibited (p<0.01), by treatment with 1 and 10 μM dopamine, with no differences between either dose as early as 1 h after treatment. The percentage of insulin-positive cells in the islets decreased significantly (p<0.01) after 1 h of treatment up to 12 h. The proliferation rate of insulin-positive cells in the islets decreased significantly (p<0.01) following treatment with dopamine. Apoptosis in pancreatic islets and beta cells was increased by treatment with 1 and 10 μM dopamine along 12 h. In conclusion, these results suggest that dopamine could modulate the proliferation and apoptosis of pancreatic beta cells and that dopamine may be involved in the maintenance of pancreatic islets.     

Inflammation of the hypothalamus leads to defective pancreatic islet function

Type 2 diabetes mellitus results from the complex association of insulin resistance and pancreatic β-cell failure. Obesity is the main risk factor for type 2 diabetes mellitus, and recent studies have shown that, in diet-induced obesity, the hypothalamus becomes inflamed and dysfunctional, resulting in the loss of the perfect coupling between caloric intake and energy expenditure. Because pancreatic β-cell function is, in part, under the control of the autonomic nervous system, we evaluated the role of hypothalamic inflammation in pancreatic islet function. In diet-induced obesity, the earliest markers of hypothalamic inflammation are present at 8 weeks after the beginning of the high fat diet; similarly, the loss of the first phase of insulin secretion is detected at the same time point and is restored following sympathectomy. Intracerebroventricular injection of a low dose of tumor necrosis factor α leads to a dysfunctional increase in insulin secretion and activates the expression of a number of markers of apoptosis in pancreatic islets. In addition, the injection of stearic acid intracerebroventricularly, which leads to hypothalamic inflammation through the activation of tau-like receptor-4 and endoplasmic reticulum stress, produces an impairment of insulin secretion, accompanied by increased expression of markers of apoptosis. The defective insulin secretion, in this case, is partially dependent on sympathetic signal-induced peroxisome proliferator receptor-γ coactivator Δα and uncoupling protein-2 expression and is restored after sympathectomy or following PGC1α expression inhibition by an antisense oligonucleotide. Thus, the autonomic signals generated in concert with hypothalamic inflammation can impair pancreatic islet function, a phenomenon that may explain the early link between obesity and defective insulin secretion.     

Glucose‐stimulated insulin release: Parallel perifusion studies of free and hydrogel encapsulated human pancreatic islets

To explore the effects immune‐isolating encapsulation has on the insulin secretion of pancreatic islets and to improve our ability to quantitatively describe the glucose‐stimulated insulin release (GSIR) of pancreatic islets, we conducted dynamic perifusion experiments with isolated human islets. Free (unencapsulated) and hydrogel encapsulated islets were perifused, in parallel, using an automated multi‐channel system that allows sample collection with high temporal resolution. Results indicated that free human islets secrete less insulin per unit mass or islet equivalent (IEQ) than murine islets and with a less pronounced first‐phase peak. While small microcapsules (d = 700 µm) caused only a slightly delayed and blunted first‐phase insulin response compared to unencapsulated islets, larger capsules (d = 1,800 µm) completely blunted the first‐phase peak and decreased the total amount of insulin released. Experimentally obtained insulin time‐profiles were fitted with our complex insulin secretion computational model. This allowed further fine‐tuning of the hormone‐release parameters of this model, which was implemented in COMSOL Multiphysics to couple hormone secretion and nutrient consumption kinetics with diffusive and convective transport. The results of these GSIR experiments, which were also supported by computational modeling, indicate that larger capsules unavoidably lead to dampening of the first‐phase insulin response and to a sustained‐release type insulin secretion that can only slowly respond to changes in glucose concentration. Bioartificial pancreas type devices can provide long‐term and physiologically desirable solutions only if immunoisolation and biocompatibility considerations are integrated with optimized nutrient diffusion and insulin release characteristics by design.     

INGAP-related pentadecapeptide: its modulatory effect upon insulin secretion

We examined the effects of a pentadecapeptide having the 104-118 aminoacid sequence of islet neogenesis-associated protein (INGAP-PP) on insulin secretion, and the morphological characteristics of adult and neonatal pancreatic rat islets cultured in RPMI and 10 mM glucose for 4 days, with or without different INGAP-PP concentrations (0.1-100 mug/ml). A scrambled 15 aminoacid peptide was used as control for the specificity of INGAP-PP effect. Cultured neonatal and adult islets released insulin in response to glucose (2.8-16.7 mM) in a dose-dependent manner, and to leucine and arginine (10 mM). In all cases, the response was greater in adult islets. INGAP-PP added to the culture medium significantly enhanced glucose- and aminoacid-induced insulin release in both adult and newborn rats; however, no changes were observed with the scrambled peptide. Similar results were obtained incubating freshly isolated adult rat islets with INGAP-PP. Whereas INGAP-PP did not induce significant changes in islet survival rate or proportion/number of islet cells, it increased significantly beta-cell size. This first demonstration of the enhancing effect of INGAP-PP on the beta-cell secretory response of adult and newborn islets opens a new avenue to study its production mechanism and potential use to increase the secretory capacity of endogenous islets in intact animals or of islets preserved for future transplants.     

Effect of annexin I on insulin secretion through surface binding sites in rat pancreatic islets

This study investigates the effect of extracellular annexin I (Anx I) on regulating insulin secretion in isolated rat pancreatic islets. Results show that Anx I stimulates insulin release in pancreatic islets regardless of the presence or absence of extracellular Ca2+. In particular, confocal microscopy shows that Anx I binds to the surface of islet cells in the absence of extracellular Ca2+. However, insulin secretion through Anx I significantly decreases in trypsin-treated islets. Likewise, there is minimal binding of Anx I to the surface of trypsin-treated islets. Anti-Anx I polyclonal antibody also inhibits the stimulating effect of Anx I on insulin secretion. These results indicate that Anx I is capable of binding to the cell surface receptor, in order to regulate the stimulation of insulin release in rat pancreatic islets.